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Old world alphaviruses, such as Ross River virus (RRV), cause debilitating arthralgia during acute and chronic stages of the disease. RRV-induced cartilage degradation has been implicated as a cause of joint pain felt by RRV patients. Chondrocytes are a major cell type of cartilage and are involved in the production and maintenance of the cartilage matrix. It is thought that these cells may play a vital role in RRV disease pathogenesis. In this study, we used RNA-sequencing (RNA-Seq) to examine the transcriptomes of RRV-infected and bystander chondrocytes in the same environment. RRV containing green fluorescent protein (GFP) allowed for the separation of RRV-infected (GFP+) and bystander uninfected cells (GFP−). We found that whereas GFP+ and GFP− populations commonly presented similar gene expression profiles during infection, there were also unique signatures. For example, RIMS2 and FOXJ1 were unique to GFP+ cells, whilst Aim2 and CCL8 were only found in bystander chondrocytes. This indicates that careful selection of potential therapeutic targets is important to minimise adverse effects to the neighbouring uninfected cell populations. Our study serves as a resource to provide more information about the pathways and responses elicited by RRV in cells which are both infected and stimulated because of neighbouring infected cells.

Introduction: Mutations and misfolding of membrane proteins are associated with various disorders, hence they make suitable targets in proteomic studies. However, extraction of membrane proteins is challenging due to their low abundance, stability, and susceptibility to protease degradation. Given the limitations in existing protocols for membrane protein extraction, the aim of this investigation was to develop a protocol for a high yield of membrane proteins for isolated Natural Killer (NK) cells. This will facilitate genetic analysis of membrane proteins known as transient receptor potential melastatin 3 (TRPM3) ion channels in myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) research.Methods: Two protocols, internally identified as Protocol 1 and 2, were adapted and optimized for high yield protein extraction. Protocol 1 utilized ultrasonic and salt precipitation, while Protocol 2 implemented a detergent and chloroform/methanol approach. Protein concentrations were determined by the Pierce Bicinchoninic Acid (BCA) and the Bio-Rad DC (detergent compatible) protein assays according to manufacturer’s recommendation. Using Protocol 2, protein samples were extracted from NK cells of n = 6 healthy controls (HC) and n = 4 ME/CFS patients. In silico tryptic digest and enhanced signature peptide (ESP) predictor were used to predict high-responding TRPM3 tryptic peptides. Trypsin in-gel digestion was performed on protein samples loaded on SDS-PAGE gels (excised at 150–200 kDa). A liquid chromatography-multiple reaction monitoring (LC-MRM) method was optimized and used to evaluate the detectability of TRPM3 n = 5 proteotypic peptides in extracted protein samples.Results: The detergent-based protocol protein yield was significantly higher (p < 0.05) compared with the ultrasonic-based protocol. The Pierce BCA protein assay showed more reproducibility and compatibility compared to the Bio-Rad DC protein assay. Two high-responding tryptic peptides (GANASAPDQLSLALAWNR and QAILFPNEEPSWK) for TRPM3 were detectable in n = 10 extracted protein samples from NK cells isolated from HC and ME/CFS patients.Conclusion: A method was optimized for high yield protein extraction from human NK cells and for the first time TRPM3 proteotypic peptides were detected using LC-MRM. This new method provides for future research to assess membrane protein structural and functional relationships, particularly to facilitate proteomic investigation of TRPM3 ion channel isoforms in NK cells in both health and disease states, such as ME/CFS.

Ross River virus (RRV) is a mosquito-borne zoonotic arbovirus associated with high public health and economic burdens across Australia, but particularly in South East Queensland (SEQ). Despite this high burden, humans are considered incidental hosts. Transmission of RRV is maintained among mosquitoes and many nonhuman vertebrate reservoir hosts, although the relative contributions of each of these hosts are unclear. To clarify the importance of a range of vertebrates in RRV transmission in SEQ, a total of 595 serum samples from 31 species were examined for RRV exposure using a gold-standard plaque reduction neutralization test. Data were analyzed statistically using generalized linear models and a coefficient inference tree, and spatially. RRV exposure was highly variable between and within species groups. Critically, species group ("placental mammal," "marsupial," and "bird"), which has previously been used as a proxy for reservoir hosts, was a poor correlate for exposure. Instead, we found that generalized "diet" and greater "body mass" were most strongly correlated with seropositivity. We also identified significant differences in seropositivity between the two major possum species (ringtail possums and brushtail possums), which are ecologically and taxonomically different. Finally, we identified distinct hotspots and coldspots of seropositivity in nonhuman vertebrates, which correlated with human notification data. This is the largest diversity of species tested for RRV in a single study to date. The analysis methods within this study provide a framework for analyzing serological data in combination with species traits for other zoonotic disease, but more specifically for RRV highlight areas to target further public health research and surveillance effort.

Up until recently, Australia was considered free of Leishmania due to the absence of phlebotomine sandfly species (Diptera: Phlebotominae) known to transmit Leishmania parasites in other parts of the world. The discovery of Leishmania (Mundinia) macropodum (Kinetoplastida: Trypanosomatidae) in Northern Australia sparked questions as to the existence of alternative vectors of Leishmania. This has added to the complexity of fully understanding the parasite's interaction with its vector, which is known to be very specific. Previous findings demonstrated L. macropodum infection beyond the blood meal stage in the day-biting midges Forcipomyia (Lasiohelea) Kieffer (Diptera: Ceratopogonidae) implicating them in the parasite's life cycle. Currently, there is no conclusive evidence demonstrating this suspected vector to transmit L. macropodum to a naïve host. Therefore, this research aimed to investigate the vector competency of day-biting midge F. (Lasiohelea) to transmit L. macropodum utilising a novel technology that preserves nucleic acids. Honey-soaked Flinders Technology Associates (FTA®) filter-paper cards were used to obtain saliva expectorated from biting midges while sugar-feeding. F. (Lasiohelea) were aspirated directly off macropods from a known Leishmania-transmission site and were kept in a waxed-paper container holding a honey-coated FTA® card for feeding. Insect identification and Taqman quantitative real-time PCR (qPCR) screening assays revealed L. macropodum DNA in F. (Lasiohelea) up to 7 days post field-collection, and in an unidentified biting midge, previously known as F. (Lasiohelea) sp.1. Moreover, 7/145 (4.83%) of FTA® cards were confirmed positive with L. macropodum DNA after exposure to field-collected F. (Lasiohelea). Additionally, FTA® cards were found to be a valuable surveillance tool, given the ease of use in the field and laboratory. Overall, our findings support previous reports on L. macropodum transmission by an alternative vector to phlebotomine sandflies. Further studies identifying and isolating infective L. macropodum promastigotes is necessary to resolve questions on the L. macropodum vector., Graphical abstract The graphical abstract illustrates the life cycle of Leishmania* within a phlebotomine sandfly vector. Infected macrophages containing Leishmania amastigotes are ingested when a sand fly feeds on an infected host (1). Within the abdominal midgut the amastigotes transform into replicative procyclic promastigotes, which are protected from digestive enzymes by the peritrophic membrane (PM) (2). The procyclic parasites differentiates into highly motile nectomonads to escape from a broken PM (3). Migrating towards the anterior midgut, nectomonads will attach to the microvilli of the midgut epithelium until reaching the stomodeal valve. To resume parasite replication, the nectomonad promastigotes are transformed into leptomonad promastigotes (4), which can either attach to the cuticle lining of the stomodeal valve as haptomonad promastigotes (5) or differentiate into metacyclic promastigotes (infective form) (5). The infective Leishmania form will colonise the stomodeal valve and are injected into the host during feeding such as on a blood meal or in this case a honey-coated FTA® card (6). *Note, the life cycle is described for suprapylarian Leishmania species where development is restricted to the sandfly midgut.Image 1, Highlights • 4.83% of FTA® cards were positive for Leishmania macropodum DNA after exposure to field-collected Forcipomyia (Lasiohelea). • L. macropodum DNA was detectable on cards after 10 weeks demonstrating their utility in Leishmania surveillance programs. • Real-time qPCR confirmed L. macropodum DNA in the previously implicated vector, F. (Lasiohelea) sp.1. • Our results support previous findings suggesting L. macropodum is transmitted by an alternative vector to the sandfly.

BackgroundAlphaviruses, such as Ross River (RRV) and chikungunya virus (CHIKV), cause significant global morbidity, with outbreaks of crippling joint inflammation and pain, leaving patients incapacitated for months to years. With no available vaccine or specific therapeutic for any alphaviral disease, and a growing economic and public health burden, there is a serious need for the development of specific therapies.MethodsThis study evaluated the safety and efficacy of pentosan polysulfate sodium (PPS) in subjects with RRV-induced arthralgia in a double-blind, placebo-controlled trial. Twenty subjects were randomized 2:1 to subcutaneous PPS (2 mg/kg) or placebo (sodium chloride 0.9%) twice weekly for 6 weeks. Safety evaluation included physical examination, concomitant medications, and laboratory findings. Efficacy assessments included change from baseline in joint function (hand grip strength and RAPID3) and quality of life (SF-36) at Days 15, 29, 39 and 81 after treatment initiation. Inflammatory and cartilage degradation biomarkers were exploratory endpoints.ResultsPPS was well tolerated, with a similar proportion of subjects reporting at least one treatment-emergent adverse event (TEAE) in the treatment and placebo groups. Injection site reactions were the most common TEAE and occurred more frequently in the PPS group. Dominant hand grip strength and SF-36 scores improved with PPS at all time points assessed, with hand grip strength improvement of 6.99 kg (p = 0.0189) higher than placebo at Day 15. PPS showed significant improvements versus placebo in adjusted mean relative change from baseline for RAPID3 Pain (p = 0.0197) and Total (p = 0.0101) scores at Day 15. At the conclusion of the study overall joint symptoms, assessed by RAPID3, showed near remission in 61.5% of PPS subjects versus 14.3% of placebo subjects. Additionally, PPS treatment improved COMP, CTX-II, CCL1, CXCL12, CXCL16 and CCL17 biomarker levels versus placebo.ConclusionsOverall, the improvements in strength and joint symptoms warrant further evaluation of PPS as a specific treatment for RRV-induced and other forms of arthritis.Trial registrationThis trial is registered at the Australian New Zealand Clinical Trials Registry #ACTRN12617000893303.

The complement alternative pathway (AP) is tightly regulated and changes in two important AP components, factor B (FB) and factor H (FH) are linked to severe dengue in humans. Here, a mouse model of dengue was investigated to define the changes in FB and FH and assess the utility of this model to study the role of the AP in severe dengue. Throughout the period of viremia in the AG129 IFN signalling-deficient mouse, an increase in FB and a decrease in FH was observed following dengue virus (DENV) infection, with the former only seen in a model of more severe disease associated with antibody-dependent enhancement (ADE). Terminal disease was associated with a decrease in FB and FH, with greater changes during ADE, and accompanied by increased C3 degradation consistent with complement activation. In silico analysis of NFκΒ, signal transducer and activator of transcription (STAT) and IFN-driven FB and FH promoter elements to reflect the likely impact of the lack of IFN-responses in AG129 mice, demonstrated that these elements differed markedly between human and mouse, notably with mouse FH lacking NFκΒ and key IFN-stimulated response elements (ISRE), and FB with many more NFκΒ and STAT-responsive elements than human FB. Thus, the AG129 mouse offers utility in demonstrating changes in FB and FH that, similar to humans, are associated with severe disease, but lack predicted important human-specific and IFN-dependent responses of FB and FH to DENV-infection that are likely to regulate the subtleties of the overall AP response during dengue disease in humans.

Over the last decade, an arbovirus surveillance system based on the preservation of nucleic acids (RNA/DNA) has been developed using Flinders Technology Associates (FTAⓇ) cards. Soaked in honey, FTAⓇ cards are applied in the field to detect arboviruses expectorated during mosquito sugar feeding. This technique has been shown to be inexpensive and efficient, and the implementation of this system for detecting parasites could be of international importance. As Leishmania parasites are highly prevalent in developing countries, FTAⓇ cards may offer an alternative inexpensive tool to enhance field surveillance activities for leishmaniasis. The simple approach of applying the cards in programs can substitute the necessary extensive training of personnel. In our hands, Leishmania macropodum DNA was shown to be stable on FTAⓇ cards during a 10-week time course, supporting their suitability for projects where direct access to laboratories is unobtainable and samples require storage prior to processing. This method may benefit programs in remote areas where accessibility to laboratory facilities are limited and samples need to be stored long-term.•This study found that FTA cards could be a valuable tool in the surveillance of leishmaniasis.•The method is based on the long-term preservation and detection of Leishmania DNA expectorated during insect sugar feeding.•The application of FTA cards can preclude the need to screen large samples and analysis of insect populations to provide evidence of disease transmission., Graphical abstract Image, graphical abstract

Up to 75 % of emerging human diseases are zoonoses, spread from animals to humans. Although bacteria, fungi and parasites can be causative agents, the majority of zoonotic infections are caused by viral pathogens. During the past 20 years many factors have converged to cause a dramatic resurgence or emergence of zoonotic diseases. Some of these factors include demographics, social changes, urban sprawl, changes in agricultural practices and global climate changes. In the period between 2014-2017 zoonotic viruses including ebola virus (EBOV), chikungunya virus (CHIKV), dengue virus (DENV) and zika virus (ZIKV), caused prominent outbreaks resulting in significant public health and economic burdens, especially in developing areas where these diseases are most prevalent. When a viral pathogen invades a new human host, it is the innate immune system that serves as the first line of defence. Myeloid cells are especially important to help fight viral infections, including those of zoonotic origins. However, viruses such as EBOV, CHIKV, DENV and ZIKV have evolved mechanisms that allow circumvention of the host's innate immune response, avoiding eradication and leading to severe clinical disease. Herein, the importance of myeloid cells in host defence is discussed and the mechanisms by which these viruses exploit myeloid cells are highlighted. The insights provided in this review will be invaluable for future studies looking to identify potential therapeutic targets towards the treatment of these emerging diseases.

Since its discovery in 2001, human metapneumovirus (hMPV) has been identified as an important cause of respiratory tract infection in young children, second only to the closely related respiratory syncytial virus (RSV). Clinical evidence suggests that hMPV is associated with acute exacerbations of asthma in both children and adults, and may play a role in initiating asthma development in children. Animal models have demonstrated that airway hyperresponsiveness (AHR) and inflammation are triggered following hMPV infection, and hMPV is able to persist in vivo by inhibiting innate immune responses and causing aberrant adaptive responses. In this review, we discuss the prevalence of hMPV infection in pediatric and adult populations and its potential role in asthma exacerbation. We also review recent advances made in animal models to determine immune responses following hMPV infection, and compare to what is known about RSV.

BACKGROUND.: Human metapneumovirus (hMPV) infection is implicated in exacerbations of asthma and chronic obstructive pulmonary disease (COPD). Research into the pathogenesis of infection is restricted to animal models, and information about hMPV replication and inflammatory and immune responses in human disease is limited. METHODS.: Human primary bronchial epithelial cells (PBECs) from healthy and asthmatic subjects and those with COPD were infected with hMPV, with or without glucocorticosteroid (GCS) exposure. Viral replication, inflammatory and immune responses, and apoptosis were analyzed. We also determined whether adjuvant interferon (IFN) can blunt hMPV infection in vitro and in a murine model. RESULTS.: hMPV infected human PBECs and viral replication was enhanced in cells from patients with COPD. The virus induced gene expression of IFN-stimulated gene 56 (ISG56) and IFN-β, as well as IFN-γ-inducible protein 10 (IP-10) and regulated on activation, normal T cell expressed and secreted (RANTES), and more so in cells from patients with COPD. GCS exposure enhanced hMPV replication despite increased IFN expression. Augmented virus replication associated with GCS was mediated by reduced apoptosis via induction of antiapoptotic genes. Adjuvant IFN treatment suppressed hMPV replication in PBECs and reduced hMPV viral titers and inflammation in vivo. CONCLUSIONS.: hMPV infects human PBECs, eliciting innate and inflammatory responses. Replication is enhanced by GCS and adjuvant IFN is an effective treatment, restricting virus replication and proinflammatory consequences of hMPV infections.

The Australasian Virology Society (AVS) aims to promote, support and advocate for the discipline of virology in the Australasian region. The society was incorporated in 2011 after 10 years operating as the Australian Virology Group (AVG) founded in 2001, coinciding with the inaugural biennial scientific meeting. AVS conferences aim to provide a forum for the dissemination of all aspects of virology, foster collaboration, and encourage participation by students and post-doctoral researchers. The tenth Australasian Virology Society (AVS10) scientific meeting was held on 2–5 December 2019 in Queenstown, New Zealand. This report highlights the latest research presented at the meeting, which included cutting-edge virology presented by our international plenary speakers Ana Fernandez-Sesma and Benjamin tenOever, and keynote Richard Kuhn. AVS10 honoured female pioneers in Australian virology, Lorena Brown and Barbara Coulson. We report outcomes from the AVS10 career development session on “Successfully transitioning from post-doc to lab head”, winners of best presentation awards, and the AVS gender equity policy, initiated in 2013. Plans for the 2021 meeting are underway which will celebrate the 20th anniversary of AVS where it all began, in Fraser Island, Queensland, Australia.

Infection with Ross River virus (RRV) causes debilitating polyarthritis and arthralgia in individuals. Alphaviruses are highly sensitive to type I interferon (IFN). Mutations at the conserved P3 position of the cleavage site between nonstructural protein 1 (nsP1) and nsP2 (1/2 site) modulate type I IFN induction for both RRV and Sindbis virus (SINV). We constructed and characterized RRV-T48A534V, a mutant harboring an A534V substitution in the P1 position of the 1/2 site, and compared it to parental RRV-T48 and to RRV-T48A532V, SINVI538 and SINVT538 harboring different substitutions in the same region. A534V substitution resulted in impaired processing of RRV nonstructural polyprotein and in elevated production of replicase-generated pathogen-associated molecular pattern (PAMP) RNAs that induce expression of type I IFN. Both A532V and A534V substitutions affected synthesis of viral RNAs, though the effects of these closely located mutations were drastically different affecting mostly either the viral negative-strand RNA or genomic and subgenomic RNA levels, respectively. Synthesis of PAMP RNAs was also observed for SINV replicase, and it was increased by I538T substitution. In comparison to RRV-T48, RRV-T48A534V was attenuated in vitro and in vivo. Interestingly, when type I IFN-deficient cells and type I IFN receptor-deficient mice were infected with RRV-T48 or RRV-T48A534V, differences between these viruses were no longer apparent. Compared to RRV-T48, RRV-T48A534V infection was associated with increased upregulation of type I IFN signaling proteins. We demonstrate novel mechanisms by which the A534V mutation affect viral nonstructural polyprotein processing that can impact PAMP RNA production, type I IFN induction/sensitivity, and disease., IMPORTANCE This study gives further insight into mechanisms of type I IFN modulation by the medically important alphaviruses Ross River virus (RRV) and Sindbis virus (SINV). By characterizing attenuated RRV mutants, the crucial role of amino acid residues in P1 and P3 positions (the first and third amino acid residues preceding the scissile bond) of the cleavage site between nsP1 and nsP2 regions was highlighted. The study uncovers a unique relationship between alphavirus nonstructural polyprotein processing, RNA replication, production of different types of pathogen-associated molecular pattern (PAMP) RNAs, type I IFN induction, and disease pathogenesis. This study also highlights the importance of the host innate immune response in RRV infections. The viral determinants of type I IFN modulation provide potential drug targets for clinical treatment of alphaviral disease and offer new approaches for rational attenuation of alphaviruses for construction of vaccine candidates.

In this chapter, we will review how viruses can be used to positively affect joints and cartilage of their hosts. Many viruses are arthrogenic, and cause persistent and debilitating arthritis. Even those viruses that are not typically arthrogenic can also cause bone lesions as secondary pathogenesis. Some of these foes include members of the alphaviruses, like chikungunya and Ross River viruses, the rubiviruses, such as rubella, and erythoparvoviruses, like parvovirus B19. Some more uncommon viruses, which can occasionally have detrimental effects on their hosts’ joints, include herpes simplex virus, varicella zoster, mumps, human cytomegalovirus, avian orthoreovirus, and caprine arthritis-encephalitis virus. Despite some viruses having negative impacts on cartilage and joints, others have been used as an effective means of gene therapy for bone and cartilage repair. We will take an in-depth look at the current therapeutic strategies for treating arthritis using various viral vectors.

Arthritogenic alphavirus infections often result in debilitating musculoskeletal disorders that affect the joints, muscle, and bone. In order to evaluate the infection profile of primary human skeletal muscle and chondrocyte cells to Ross River virus (RRV) in vitro, cells were infected at a multiplicity of infection (MOI) of 1 over a period of two days. Viral titers were determined by plaque assay and cytokine expression by Bio-Plex® assays using the supernatants harvested. Gene expression studies were conducted using total RNA isolated from cells. Firstly, we show that RRV RNA is detected in chondrocytes from infected mice in vivo. Both human primary skeletal muscle and chondrocyte cells are able to support productive RRV infection in vitro. We also report the production of soluble host factors including the upregulation of heparanase (HPSE) and inflammatory host factors such as interleukin-6 (IL-6), monocyte chemoattractant protein 1 (MCP-1), RANTES (regulated on activation, normal T cell expressed and secreted), interferon gamma (IFN-γ), and tumor necrosis factor alpha (TNF-α), which are also present during clinical disease in humans. Our study is the first to demonstrate that human chondrocyte cells are permissive to RRV infection, support the production of infectious virus, and produce soluble factors including HPSE, which may contribute to joint degradation and the pathogenesis of disease.

Re-emergence of chikungunya virus, a mosquito-transmitted pathogen, is of serious public health concern. In the past 15 years, after decades of infrequent, sporadic outbreaks, the virus has caused major epidemic outbreaks in Africa, Asia, the Indian Ocean, and more recently the Caribbean and the Americas. Chikungunya virus is mainly transmitted by Aedes aegypti mosquitoes in tropical and subtropical regions, but the potential exists for further spread because of genetic adaptation of the virus to Aedes albopictus, a species that thrives in temperate regions. Chikungunya virus represents a substantial health burden to affected populations, with symptoms that include severe joint and muscle pain, rashes, and fever, as well as prolonged periods of disability in some patients. The inflammatory response coincides with raised levels of immune mediators and infiltration of immune cells into infected joints and surrounding tissues. Animal models have provided insights into disease pathology and immune responses. Although host innate and adaptive responses have a role in viral clearance and protection, they can also contribute to virus-induced immune pathology. Understanding the mechanisms of host immune responses is essential for the development of treatments and vaccines. Inhibitory compounds targeting key inflammatory pathways, as well as attenuated virus vaccines, have shown some success in animal models, including an attenuated vaccine strain based on an isolate from La Reunion incorporating an internal ribosome entry sequence that prevents the virus from infecting mosquitoes and a vaccine based on virus-like particles expressing envelope proteins. However, immune correlates of protection, as well as the safety of prophylactic and therapeutic candidates, are important to consider for their application in chikungunya infections. In this Review, we provide an update on chikungunya virus with regard to its epidemiology, molecular virology, virus-host interactions, immunological responses, animal models, and potential antiviral therapies and vaccines.

Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-β promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1 −/− > IRF3/7 −/− > IRF3 −/− , but not IRF7 −/− , mice compared with wild-type mice. IFN-β and IFN-λ2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-λ2/3 was greater in IRF3/7 −/− mice at 5 days after infection. IRF7 −/− and IRF3/7 −/− mice presented with airway eosinophilia, whereas only IRF3/7 −/− mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1 −/− mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-β, but not IFN-λ2/3, produced via an IPS-1–IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-α/β response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence.

Human respiratory syncytial virus (hRSV) is a common respiratory virus that is usually no cause for alarm. Symptoms of hRSV usually resemble those of the common cold and can go undiagnosed. However, infants as well as the elderly are at risk for developing severe cases, which can lead to high morbidity and mortality rates especially if there are underlying health issues. Despite many years of effort, no vaccine or specific treatments exist and RSV is still the leading cause of infant hospitalizations worldwide. Here, we describe methods to infect two widely used small animal models: laboratory mice and cotton rats.

The majority of medical advances have been made using animals. Studies using mouse models of chikungunya-induced disease have proven invaluable for dissecting the intricate nature of the immune response to this viral infection and identifying potential targets for the development of treatment strategies. Herein we describe the common mouse models used to research the pathobiology of chikungunya virus infection to date.

With an expanding geographical range and no specific treatments, human arthritogenic alphaviral disease poses a significant problem worldwide. Previous in vitro work with Ross River virus (RRV) demonstrated that alphaviral N-linked glycosylation contributes to type I IFN (IFN-αβ) induction in myeloid dendritic cells. This study further evaluated the role of alphaviral N-linked glycans in vivo, assessing the effect of glycosylation on pathogenesis in a mouse model of RRV-induced disease and on viral infection and dissemination in a common mosquito vector, Aedes vigilax. A viral mutant lacking the E1-141 glycosylation site was attenuated for virus-induced disease, with reduced myositis and higher levels of IFN-γ induction at peak disease contributing to improved viral clearance, suggesting that glycosylation of the E1 glycoprotein plays a major role in the pathogenesis of RRV. Interestingly, RRV lacking E2-200 glycan had significantly reduced replication in the mosquito vector A. vigilax, whereas loss of either of the E1 or E2-262 glycans had little effect on the competence of the mosquito vector. Overall, these results indicate that glycosylation of the E1 and E2 glycoproteins of RRV provides important determinants of viral virulence and immunopathology in the mammalian host and replication in the mosquito vector.

The majority of medical advances have been made using animals. Studies using mouse models of chikungunya-induced disease have proven invaluable for dissecting the intricate nature of the immune response to this viral infection and identifying potential targets for the development of treatment strategies. Herein we describe the common mouse models used to research the pathobiology of chikungunya virus infection to date.

Human respiratory syncytial virus (hRSV) is a common respiratory virus that is usually no cause for alarm. Symptoms of hRSV usually resemble those of the common cold and can go undiagnosed. However, infants as well as the elderly are at risk for developing severe cases, which can lead to high morbidity and mortality rates especially if there are underlying health issues. Despite many years of effort, no vaccine or specific treatments exist and RSV is still the leading cause of infant hospitalizations worldwide. Here, we describe methods to infect two widely used small animal models: laboratory mice and cotton rats.

Even though ferrets are one of the principal animal models for influenza pathogenesis, the lack of suitable immunological reagents has so far limited their use in host response studies. Using recently established real-time PCR assays for a panel of ferret cytokines, we analyzed the local ferret immune response to human influenza isolates of the H1N1 and H3N2 subtypes that varied in their virulence. We observed that the severity of clinical signs correlated with gross- and histopathological changes in the lungs and was subtype-independent. Strains causing a mild disease were associated with a strong and rapid innate response and upregulation of IL-8, while severe infections were characterized by a lesser induction of type I and II interferons and strong IL-6 upregulation. These findings suggest that more virulent strains may interfere more efficiently with the host response at early disease stages.

Chikungunya virus (CHIKV) is a reemerging, ordinarily mosquito-transmitted, alphavirus that occasionally produces hemorrhagic manifestations, such as nose bleed and bleeding gums, in human patients. Interferon response factor 3 and 7 deficient (IRF3/7-/-) mice, which are deficient for interferon α/β responses, reliably develop hemorrhagic manifestations after CHIKV infection. Here we show that infectious virus was present in the oral cavity of CHIKV infected IRF3/7-/- mice, likely due to hemorrhagic lesions in the olfactory epithelium that allow egress of infected blood into the nasal, and subsequently, oral cavities. In addition. IRF3/7-/- mice were more susceptible to infection with CHIKV via intranasal and oral routes, with IRF3/7-/- mice also able to transmit virus mouse-to-mouse without an arthropod vector. Cynomolgus macaques often show bleeding gums after CHIKV infection, and analysis of saliva from several infected monkeys also revealed the presence of viral RNA and infectious virus. Furthermore, saliva samples collected from several acute CHIKV patients with hemorrhagic manifestations were found to contain viral RNA and infectious virus. Oral fluids can therefore be infectious during acute CHIKV infections, likely due to hemorrhagic manifestations in the oral/nasal cavities. Refereed/Peer-reviewed

Canine distemper virus (CDV), a member of the Morbillivirus genus that also includes measles virus, frequently causes neurologic complications, but the routes and timing of CDV invasion of the central nervous system (CNS) are poorly understood. To characterize these events, we cloned and sequenced the genome of a neurovirulent CDV (strain A75/17) and produced an infectious cDNA that expresses the green fluorescent protein. This virus fully retained its virulence in ferrets: the course and signs of disease were equivalent to those of the parental isolate. We observed CNS invasion through two distinct pathways: anterogradely via the olfactory nerve and hematogenously through the choroid plexus and cerebral blood vessels. CNS invasion only occurred after massive infection of the lymphatic system and spread to the epithelial cells throughout the body. While at early time points, mostly immune and endothelial cells were infected, the virus later spread to glial cells and neurons. Together, the results suggest similarities in the timing, target cells, and CNS invasion routes of CDV, members of the Morbillivirus genus, and even other neurovirulent paramyxoviruses like Nipah and mumps viruses.

Mammalian reoviruses exhibit a propensity to replicate in transformed cells. It is currently believed that the interferon-inducible RNA-dependent protein kinase (PKR), an intracellular host-cell resistance factor that is inhibited by an activatedRas-dependent pathway in transformed cells, is responsible for this discrimination. In the present study, reovirus isolates differing in their sensitivity to interferon were obtained by chemical mutagenesis, and examined for their replicative properties in parental andRas-transformed mouse NIH-3T3 cells. It was observed that most isolates can bypass resistance mechanisms of parental cells at high m.o.i., and that there is a correlation between the ability to discriminate between transformed and parental cells, and interferon sensitivity. Most interestingly, an interferon-hypersensitive mutant virus was more dependent onRasactivation than any other viral isolate. Altogether, this suggests that optimal reovirus isolates could be selected to attack tumour cells depending on the nature of the alterations in interferon-inducible pathways found in these cells.

Human respiratory syncytial virus (RSV) is a major cause of morbidity and severe lower respiratory tract disease in the elderly and very young, with some infants developing bronchiolitis, recurrent wheezing, and asthma following infection. Previous studies in humans and animal models have shown that vaccination with formalin-inactivated RSV (FI-RSV) leads to prominent airway eosinophilic inflammation following RSV challenge; however, the roles of pulmonary eosinophilia in the antiviral response and in disease pathogenesis are inadequately understood. In vivo studies in mice with eotaxin and/or interleukin 5 (IL-5) deficiency showed that FI-RSV vaccination did not lead to enhanced pulmonary disease, where following challenge there were reduced pulmonary eosinophilia, inflammation, Th2-type cytokine responses, and altered chemokine (TARC and CCL17) responses. In contrast to wild-type mice, RSV was recovered at high titers from the lungs of eotaxin- and/or IL-5-deficient mice. Adoptive transfer of eosinophils to FI-RSV-immunized eotaxin- and IL-5-deficient (double-deficient) mice challenged with RSV was associated with potent viral clearance that was mediated at least partly through nitric oxide. These studies show that pulmonary eosinophilia has dual outcomes: one linked to RSV-induced airway inflammation and pulmonary pathology and one with innate features that contribute to a reduction in the viral load. IMPORTANCE This study is critical to understanding the mechanisms attributable to RSV vaccine-enhanced disease. This study addresses the hypothesis that IL-5 and eotaxin are critical in pulmonary eosinophil response related to FI-RSV vaccine-enhanced disease. The findings suggest that in addition to mediating tissue pathology, eosinophils within a Th2 environment also have antiviral activity.

Part of the Togaviridae family, alphaviruses are arthropod-borne viruses that are widely distributed throughout the globe. Alphaviruses are able to infect a variety of vertebrate hosts, but in humans, infection can result in extensive morbidity and mortality. Symptomatic infection can manifest as fever, an erythematous rash and/or significant inflammatory pathologies such as arthritis and encephalitis. Recent overwhelming outbreaks of alphaviral disease have highlighted the void in our understanding of alphavirus pathogenesis and the re-emergence of alphaviruses has given new impetus to anti-alphaviral drug design. In this review, the development of viable mouse models of Old Word and New World alphaviruses is examined. How mouse models that best replicate human disease have been used to elucidate the immunopathology of alphavirus pathogenesis and trial novel therapeutic discoveries is also discussed.

Foroderm is a new cutaneous delivery technology that uses high-aspect ratio, cylindrical silica microparticles, that are massaged into the skin using a 3D-printed microtextured applicator, in order to deliver payloads across the epidermis. Herein we show that this technology is effective for delivery of a non-adjuvanted, inactivated, whole-virus chikungunya virus vaccine in mice, with minimal post-vaccination skin reactions. A single topical Foroderm-based vaccination induced T cell, Th1 cytokine and antibody responses, which provided complete protection against viraemia and disease after challenge with chikungunya virus. Foroderm vaccination was shown to deliver fluorescent, virus-sized beads across the epidermis, with beads subsequently detected in draining lymph nodes. Foroderm vaccination also stimulated the egress of MHC II+ antigen presenting cells from the skin. Foroderm thus has potential as a simple, cheap, effective, generic, needle-free technology for topical delivery of vaccines. Refereed/Peer-reviewed

In the context of viral infections, autophagy induction can be beneficial or inhibitory. Within the Paramyxoviridae family, only morbilliviruses have been investigated and are reported to induce autophagy. Here we show that morbilliviruses rapidly induce autophagy and require this induction for efficient cell-to-cell spread. Coexpression of both glycoproteins in cells expressing one of the cellular receptors was required for autophagy induction, and LC3 punctum formation, indicative of autophagy, was mainly observed in syncytia. A similar correlation between syncytium formation and autophagy induction was also observed for other paramyxovirus glycoproteins, suggesting that membrane fusion-mediated autophagy may be common among paramyxoviruses and possibly other enveloped viruses.

Human metapneumovirus (hMPV) is a leading cause of respiratory tract disease in children and is associated with acute bronchiolitis, pneumonia, and asthma exacerbations, yet the mechanisms by which the host immune response to hMPV is regulated are poorly understood. By using gene-deleted neonatal mice, we examined the contributions of the innate receptor signaling molecules interferon (IFN)-β promoter stimulator 1 (IPS-1), IFN regulatory factor (IRF) 3, and IRF7. Viral load in the lungs was markedly greater in IPS-1 −/− > IRF3/7 −/− > IRF3 −/− , but not IRF7 −/− , mice compared with wild-type mice. IFN-β and IFN-λ2/3 (IL-28A/B) production was attenuated in the bronchoalveolar lavage fluid in all factor-deficient mice compared with wild-type mice at 1 day after infection, although IFN-λ2/3 was greater in IRF3/7 −/− mice at 5 days after infection. IRF7 −/− and IRF3/7 −/− mice presented with airway eosinophilia, whereas only IRF3/7 −/− mice developed an exaggerated type 1 and 17 helper T-cell response, characterized by natural killer T-cell and neutrophilic inflammation. Despite having the highest viral load, IPS-1 −/− mice did not develop a proinflammatory cytokine or granulocytic response to hMPV infection. Our findings demonstrate that IFN-β, but not IFN-λ2/3, produced via an IPS-1-IRF3 signaling pathway, is important for hMPV clearance. In the absence of a robust type I IFN-α/β response, targeting the IPS-1 signaling pathway may limit the overexuberant inflammatory response that occurs as a consequence of viral persistence.

For most virus infections of the central nervous system (CNS), immune-mediated damage, the route of inoculation and death of infected cells all contribute to the pathology observed. To investigate the role of these factors in early canine distemper neuropathogenesis, we infected ferrets either intranasally or intraperitoneally with the neurovirulent canine distemper virus strain Snyder Hill. Regardless of the route of inoculation, the virus primarily targeted the olfactory bulb, brainstem, hippocampus and cerebellum, whereas only occasional foci were detected in the cortex. The infection led to widespread neuronal loss, which correlated with the clinical signs observed. Increased numbers of activated microglia, reactive gliosis and different pro-inflammatory cytokines were detected in the infected areas, suggesting that the presence and ultimate death of infected cells at early times after infection trigger strong local immune activation, despite the observed systemic immunosuppression.

The Morbillivirus hemagglutinin (H) protein mediates attachment to the target cell. To evaluate its contribution to canine distemper virus neurovirulence, we exchanged the H proteins of the wild-type strains 5804P and A75 and assessed the pathogenesis of the chimeric viruses in ferrets. Both strains are lethal to ferrets; however, 5804P causes a 2-week disease without neurological signs, whereas A75 is associated with a longer disease course and neurological involvement. We observed that both H proteins supported neuroinvasion and the subsequent development of clinical neurological signs if given enough time, demonstrating that disease duration is the main neurovirulence determinant.

A new procedure for measuring ATPase activity in which γ-33P-labeled inorganic orthophoshate is detected by addition of ammonium molybdate followed by selective adsorption of the resulting phosphomolybdate to scintillation proximity beads in the presence of cesium chloride is described. This method is shown to give accurate and reproducible results over a wide range of detection conditions and product concentrations. It requires no separation or filtration steps and is highly compatible with automated high-throughput screening. Rates of hydrolysis are easily and accurately determined over a wide range, and thus the method is useful for kinetic studies also. We show that this scintillation proximity assay is useful for the study of the E1 helicase of human papillomavirus, but it is a general procedure which could also be applied to any ATPase or other nucleotide triphosphate-hydrolyzing enzyme or any other enzyme which generates orthophosphate as a reaction product.